Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human aquaporin 3 (AQP3) gene was isolated, and its structural organization was characterized. The gene appeared to exist as a single copy in the human genome and to comprise six exons distributing over 7 kilobases. The sizes of the exons are 171, 127, 138, 119, 218, and 1035 base pairs, and those of introns are approximately 3530, 300, 350, 330, and 90 base pairs, respectively. The initiation site of transcription was identified to locate 64 base pairs upstream of the first ATG codon by primer extension analysis and ribonuclease protection assay. The 5'-flanking region has a TATA box, two Sp1 sequences, and some consensus sequences including AP2 sites. With luciferase assay, the 5'-flanking region was demonstrated to have a promoter activity, which is up-regulated 4-fold by phorbol ester. These findings about the genomic clone of human AQP3 will contribute to elucidate the molecular mechanism of transcriptional regulation of AQP3.
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PMID:Isolation of human aquaporin 3 gene. 754 93

KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5' of the initiation methionine codon. The 5'-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that approximately 4 kilobases of KDR/flk-1 5'-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp -225 to -164, -95 to -77, -77 to -60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells.
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PMID:Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor. 755 54

Monocyte-macrophage differentiation was used as a model system for studying gene regulation of the human vacuolar H(+)-ATPase (V-ATPase). We examined mRNA levels of various V-ATPase subunits during differentiation of both native monocytes and the cell line THP-1, and found that transcriptional and post-transcriptional mechanisms could account for increases in cell V-ATPase content. From nuclear runoff experiments, we found that one subunit in particular, the B2 isoform (Mr = 56,000), was amplified primarily by transcriptional means. We have begun to examine the structure of the B2 subunit promoter region. Isolation and sequencing of the first exon and 5'-flanking region of this gene reveal a TATA-less promoter with a high G + C content. Primer extension and ribonuclease protection analyses indicate a single major transcriptional start site. We transfected promoter-luciferase reporter plasmids into THP-1 cells to define sequences that mediate transcriptional control during monocyte differentiation. We found that sequences downstream from the transcriptional start site were sufficient to confer increased expression during THP-1 differentiation. DNase I footprinting and sequence analysis revealed the existence of multiple AP2 and Sp1 binding sites in the 5'-untranslated and proximal coding regions.
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PMID:Transcriptional regulation of the vacuolar H(+)-ATPase B2 subunit gene in differentiating THP-1 cells. 770 73

To study the differential expression of the murine VLA-4 (alpha 4 beta 1) integrin, the 5'-flanking region of the gene for the alpha subunit (alpha 4m) was isolated and a cDNA for alpha 4m was obtained with reverse transcriptase polymerase chain reaction (RT-PCR). The cDNA sequence contained a difference in the signal peptide region compared to the previously described cDNA (Neuhaus et al., 1991). As a consequence, another start codon is predicted, resulting in a decrease in size of the signal peptide. This was confirmed by genomic sequencing. The promoter region was delimited by ribonuclease protection assay (RPA) and transfection experiments fusing 5'-upstream fragments to the luciferase gene. A fragment extending from -936 to +221 was capable of controlling the expected cell-type-specific expression. Sequence comparison of the mouse alpha 4m promoter region with the human alpha 4h promoter revealed little homology. Like most integrin subunits, alpha 4m lacks TATA anc CCAAT boxes. Putative recognition sites for DNA-binding nuclear factors (AP1, AP2, Sp1, and PU1) were identified. The characterization of the promoter region and further identification of the transcription regulatory elements should provide insight in the regulation of alpha 4m integrin gene expression.
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PMID:Cloning and characterization of the promoter region of the murine alpha-4 integrin subunit. 777 55

The GnRH receptor (GnRH-R) is a cell surface, G protein-coupled receptor that is highly expressed in pituitary gonadotropes. Activation of the receptor by GnRH stimulates the release of FSH and LH. Pituitary GnRH-R numbers and, hence, gonadotrope responsiveness to GnRH vary under different conditions and are regulated to a large extent by GnRH itself. To study the transcriptional regulation of the GnRH-R gene, a genomic clone containing 1.2 kilobases (kb) of the 5'-flanking region of mouse GnRH-R gene was isolated and characterized. A major transcriptional start site was identified 62 nucleotides upstream of the translational start site by primer extension and ribonuclease protection analyses. The promoter region does not contain canonical TATA sequences in the appropriate location. To determine whether this putative promoter is functional, it was subcloned into a luciferase reporter plasmid (GnRH-RLuc), and its transient expression was studied in cell lines of gonadotrope (alpha T3-1) and somatolactotrope (GH3) origins as well as those of nonpituitary origin (JEG-3 and CV-1). Luciferase activity was increased in alpha T3-1 (246-fold +/- 34.5-fold; P < 0.005) compared with the promoterless vector control but was considerably lower in GH3 (41-fold +/- 3.9-fold; P < 0.005), JEG-3 (12-fold +/- 0.9-fold; P < 0.005) and CV-1 (8-fold +/- 1.3-fold) indicating that GnRH-RLuc is preferentially expressed in cells of gonadotrope origin. Furthermore, GnRH agonist stimulated luciferase activity 3.4-fold +/- 0.3-fold (P < 0.005) above basal levels in GH3 cells cotransfected with rat GnRH-R complementary DNA, indicating that the GnRH-R promoter sequence is responsive to this ligand. In summary, we have identified and partially characterized the promoter region of the mouse GnRH-R and demonstrated that the regulatory elements for tissue-specific expression as well as for GnRH regulation are present within a 1.2-kb 5'-flanking region of the mouse GnRH-R gene.
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PMID:Isolation and characterization of the 5'-flanking region of the mouse gonadotropin-releasing hormone receptor gene. 798 11

The genes for the alpha subunit of inhibin and for the nonapeptide hormone oxytocin are both expressed in the granulosa cells of the ruminant follicle as well as in the Sertoli cells of the ruminant testis. Northern hybridization of mRNA from both ovary and testis indicate that in both gonads the expression of the two genes is inversely regulated. In the luteinizing granulosa cells, in vitro as in vivo, the alpha-inhibin gene is down-regulated when the oxytocin gene is up-regulated. In the Sertoli cells of the bull and sheep testis, the situation is similar, with the alpha-inhibin gene being up-regulated in the prepubertal gonad and down-regulated concomitantly with an up-regulation of the oxytocin gene in early puberty. The gene for the bovine alpha-inhibin subunit was cloned and characterized. Assessment of transcriptional initiation by primer extension and ribonuclease protection assays showed that several different sites were used in both granulosa cells and testis. Transient transfection of primary bovine granulosa cells with alpha-inhibin/luciferase gene constructs indicated that a major promoter element resided in the region -178 to -245 respective to the methionine start codon of translation, a region that contains a cAMP response element. The ability of forskolin to up-regulate the transcription of transfected gene constructs also depended on the integrity of this region. In contrast, transfection of TM4 cells led to transcriptional initiation from an unusual site in the alpha-inhibin gene and to a lack of forskolin regulation. Comparison of the alpha-inhibin and oxytocin genes indicates that although both can be up-regulated by FSH or by forskolin within the same cells, different mechanisms of signal transduction are involved to explain the temporal differences in expression. Together the results indicate that a differentiation step occurring in Sertoli cells at early puberty and in granulosa cells at luteinization involves comparable regulation of genes through the sequential action of different cAMP-linked transcription factors.
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PMID:Structure of the alpha-inhibin gene and its regulation in the ruminant gonad: inverse relationship to oxytocin gene expression. 814 57

Cytosolic phospholipase A2 (cPLA2) releases arachidonic acid from membrane phospholipids and is believed to be the rate-limiting enzyme in the arachidonic acid pathway. We report herein the isolation of a 3 kb fragment of rodent genomic DNA containing part of the first intron, the first exon and 5'-flanking sequence. The start site of transcription was mapped by 5'-rapid amplification of cDNA ends and corroborated by ribonuclease protection assay. The gene has a TATAless promoter with no classical Sp1 binding sites or initiator element. A microsatellite series of CA repeats was noted in the 5'-flanking region of both the rodent and human promoters. Deletion constructs have been analysed for luciferase activity and confirmed promoter activity.
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PMID:Isolation of promoter for cytosolic phospholipase A2 (cPLA2). 814 85

The interleukin-2 (IL-2) receptor gamma chain is an essential component of high and intermediate affinity IL-2 receptors (IL-2Rs), playing critical roles for ligand binding and internalization. We report here the isolation and characterization of the genomic locus for human IL-2R gamma, which, like IL-2R beta, is a member of the cytokine receptor superfamily. The IL-2R gamma gene is composed of eight exons and seven introns and spans approximately 4.2 kilobases. Analogous to the IL-2R beta gene, the two pairs of conserved cysteines typical of cytokine receptor superfamily proteins are located in adjacent exons, and the conserved WSXWS motif is located in the exon preceding the one that encodes the transmembrane domain and a small part of the cytoplasmic domain. In each gene, the remainder of the cytoplasmic domain is encoded by the final two exons. Southern blot analysis suggests that IL-2R gamma is encoded by a single copy gene. Cross-hybridizing sequences were detected in DNA derived from a number of other mammalian species but not from yeast. Primer extension analysis and ribonuclease protection assays revealed that there are three principal transcription initiation sites located 32-38 nucleotides 5' to the translation initiation AUG codon. These sites are upstream of the 5' end of the published IL-2R gamma cDNA sequence. The region 5' to the transcription initiation sites exhibited promoter activity when cloned upstream of the luciferase reporter gene. With this study, the organization of the genes encoding all three chains (alpha, beta, and gamma) of the IL-2 receptor has been determined and promoters for each identified.
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PMID:Characterization of the human interleukin-2 receptor gamma chain gene. 851 92

Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen-regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells.
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PMID:A human gene encoding diazepam-binding inhibitor/acy1-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP. 863 49

Recently, a distinct family of G protein-coupled receptors has been cloned that mediates the biological effects of melatonin. Of two sub-types cloned from mammals (Mel1a and Mel1b), the Mel1a receptor appears to mediate the circadian and reproductive effects of the hormone. We now report the cloning, characterization, and expression of the gene encoding the Mel1a receptor in mice. The receptor gene is composed of two exons, separated by an intron of greater than 13 kilobases. Exon 1 encodes the entire 5'-untranslated region and the coding region through the first cytoplasmic loop. Exon 2 encodes the rest of the coding region and the entire 3'-untranslated region. 5'-Rapid amplification of complementary DNA ends and ribonuclease protection analyses show that the major transcription start site is 103 nucleotides upstream of the translation start codon. Sequence analysis of 1.1 kilobases of the 5'-flanking region reveals that it does not contain TATA or CAAT boxes. The 5'-flanking region drives luciferase expression 114-fold over basal levels in a murine retinal cell line that endogenously expresses the Mel1a receptor. The mouse receptor binds 2-[125]iodomelatonin with high affinity (K(d) = 55.6 pM) when expressed transiently in COS-7 cells. In situ hybridization studies establish that Mel1a receptor messenger RNA is expressed in the hypothalamic suprachiasmatic nuclei and hypophyseal pars tuberalis, presumed sites of the circadian and some of reproductive actions of melatonin, respectively. These results provide information on Mel1a receptor gene structure essential for designing transgenic and gene knock-out studies and analyzing the transcriptional regulation of receptor gene expression.
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PMID:Structure, characterization, and expression of the gene encoding the mouse Mel1a melatonin receptor. 875 76


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